Prospective evaluation of the clinical usefulness of an antigen-specific assay (MAIPA) in idiopathic thrombocytopenic purpura and other immune thrombocytopenias.

The diagnosis of idiopathic immune thrombocytopenia remains a clinical diagnosis based on the exclusion of other causes of immune and nonimmune thrombocytopenia. Measurement of platelet-associated Ig (PAIg), while sensitive, is nonspecific for the diagnosis of immune thrombocytopenia. Published experience of antigen capture assays (including monoclonal antibody immobilization of platelet antigens or MAIPA) suggest a high sensitivity and specificity (70% to 80%) in selected groups of patients. In a prospective evaluation of 158 patients with thrombocytopenia from all causes, we report a sensitivity of 51% and specificity of 80% for direct MAIPA assays. MAIPA was considerably better in discriminating immune from nonimmune thrombocytopenia than two assays of PAIgG. Antiplatelet antibodies detected by MAIPA were more frequently directed against the glycoprotein (GP) IIb/IIIa than the GP Ib/IX complex. Our experience suggests that MAIPA assays are useful in the laboratory assessment of thrombocytopenia, should be performed before therapy, and that some patients with 'nonimmune' thrombocytopenia may have genuine antiplatelet antibodies.

The molecular mechanism of platelet adhesion.

One of the most primitive of host-defence mechanisms is haemostasis, the ability to control blood loss. In response to vascular trauma, platelets rapidly adhere to the exposed subendothelial matrix, a process that ultimately results in the sealing of the vessel by a plug of platelets stabilised by fibrin. Paradoxically, it is the same cascade of events that leads to thrombosis and vessel occlusion, resulting in heart attack and stroke. The molecular events involved in platelet adhesion have therefore been the subject of intense investigation. In all but the largest blood vessels, the initial contact adhesion of platelets is mediated by subendothelial matrix bound von Willebrand Factor (vWF) and a specific vWF receptor on platelets, the glycoprotein (GP) Ib-V-IX complex. Our understanding of this process arose from analysis of two congenital bleeding disorders, von Willebrand's disease and the Bernard-Soulier syndrome, in which vWF or the GP Ib-V-IX, respectively, are either absent or dysfunctional. This overview discusses our current molecular understanding of platelet adhesion and how engagement of vWF by the GP Ib-V-IX complex on platelets initiates the subsequent events in platelet activation leading to either haemostasis or thrombosis.

Identification of aspartic acid 514 through glutamic acid 542 as a glycoprotein Ib-IX complex receptor recognition sequence in von Willebrand factor. Mechanism of modulation of von Willebrand factor by ristocetin and botrocetin.

As the first step in hemostasis, the binding of von Willebrand factor (vWF) to the platelet membrane glycoprotein (GP) Ib-IX complex is essential for platelet adhesion at high-shear blood flow. This interaction in vivo requires the prior binding of vWF to the subendothelial matrix, a process which exposes a normally cryptic binding site on vWF for the GP Ib-IX complex. This process can be mimicked in vitro by modulators such as ristocetin or the snake venom protein botrocetin or by desialation of vWF. We have previously localized the GP Ib binding site on vWF to a monomeric dispase fragment which extends from Leu-480/Val-481 to Gly-718 in the primary sequence of mature vWF [Andrews, R. K., Gorman, J. J., Booth, W. J., Corino, G. L., Castaldi, P. A., & Berndt, M. C. (1989) Biochemistry 28, 8326-8336]. This fragment also contains a distinct binding site for botrocetin. Analysis of synthetic peptides corresponding to hydrophilic stretches of sequence within this fragment indicated that the sequence Asp-514-Glu-542 represents a major adhesive sequence involved in receptor recognition. This peptide inhibited both the ristocetin- and botrocetin-mediated binding of vWF to either platelets or purified GP Ib-IX complex (IC50 approximately 50-200 microM) as well as the asialo-vWF- and bovine vWF-dependent agglutination of platelets. Both the N- and C-terminal halves of the peptide were inhibitory but less so than the intact peptide. This peptide also inhibited botrocetin binding to vWF, suggesting that botrocetin modulates vWF-GP Ib interaction by binding in close proximity to the vWF adhesion sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of GMP-140 (P-selectin) as a circulating plasma protein.

GMP-140 is a 140-kD granule membrane protein, found in the alpha granules of platelets and the Weibel-Palade bodies of endothelial cells, that is surface expressed on cell activation and mediates neutrophil attachment. Cloning data for GMP-140 from an endothelial library predict a soluble form of the protein, the transcription message for which is also found in platelets. In this study, we report the detection by enzyme-linked immunosorbent assay of soluble GMP-140 in plasma centrifuged for 3 h at 100,000 g (to remove platelet microparticles) and confirm its identity by purification from plasma. Plasma concentrations were found to be 0.251 +/- 0.043 micrograms/ml (means +/- SD, n = 10) in normal male controls and 0.175 +/- 0.063 micrograms/ml (means +/- SD, n = 10) in normal female controls. The purified protein had an identical molecular mass (nonreduced) to platelet membrane GMP-140 (approximately 3 kD lower, reduced) and was immunoblotted by polyclonal anti-GMP-140, and the anti-GMP-140 monoclonal antibodies AK4 and AK6. Analytical gel filtration studies indicated that the plasma GMP-140 eluted as a monomer whereas detergent-free, platelet membrane GMP-140 eluted as a tetramer consistent with plasma GMP-140 lacking a transmembrane domain. Purified plasma GMP-140 bound to the same neutrophil receptor as the membrane-bound form, and when immobilized on plastic, bound neutrophils equivalently to immobilized platelet membrane GMP-140. Since it has been shown that fluid-phase GMP-140 is antiinflammatory and downregulates CD18-dependent neutrophil adhesion and respiratory burst, its presence in plasma may be of major importance in preventing the inadvertent activation of neutrophils in the circulation.

Polymorphism of platelet glycoprotein Ib associated with variability of the 85-kDa macroglycopeptide region.

Polymorphism of platelet membrane glycoprotein Ib (GP Ib) has been described in both normal and functionally abnormal platelets. In this report we have investigated the polymorphism in one Australian Caucasian family with normal platelet function in which the following phenotypes were found: father, BC; mother, CD; son, BC; and daughter, BD; thus establishing a genetic basis for this phenomenon. The daughter's phenotype BD is highly distinctive due to the double-band pattern obtained by gel analysis. Platelets from the daughter were digested with either trypsin or elastase and the GP Ib cleavage products were examined by immunoprecipitation with anti-GP Ib-IX complex monoclonal antibodies against each of the major tryptic domains. On the basis of these studies, we have determined that the GPIb polymorphism in this family resides in the 85-kDa, macroglycopeptide region of the alpha-chain of GP Ib and not in either the 45-kDa, N-terminal region of GP Ib alpha or the membrane-associated region of the complex.

Autologous bone marrow transplantation for acute myeloid leukaemia in remission: a preliminary report.

Autologous bone marrow transplantation, using unpurged cryopreserved autologous marrow, was performed on ten adult patients with acute myeloid leukaemia in remission. Seven patients were in first chemotherapy-induced remission of their disease, while three were in later remission. Patients ages ranged from 24 to 52 years, with a median of 38.5 years. Conditioning therapy consisted of oral busulphan 16 mg/kg over four days and intravenous cyclophosphamide 60 mg/kg on two days. Bone marrow cells were thawed and infused two days later. All patients showed signs of marrow engraftment, however this was delayed in comparison with patients receiving allogeneic transplants. All patients developed fever requiring antibiotic therapy and one patient died of overwhelming sepsis. Another patient died of hepatic veno-occlusive disease two months after transplant. Serious, but non-fatal, hepatic complications occurred in two other patients. One patient, transplanted in third remission, relapsed 16 months post-autograft. No other relapses have been seen, with one second remission patient remaining leukaemia-free at 24 months, and six first remission patients in continuing remission 11 to 23 (median 20) months post transplant. These encouraging results require confirmation in a randomised clinical trial comparing autologous marrow transplantation versus standard chemotherapy.

The Interaction of von Willebrand Factor and the Platelet Glycoprotein Ib-IX Complex.

The interaction between blood platelets and the vessel wall represents the initial event in the haemostatic response to vessel injury. At a molecular level, this interaction involves the platelet membrane glycoprotein (GP) Ib-IX complex, von Willebrand factor (vWF) and a component (s) of the subendothelial matrix. In recent years, the primary sequences of both the vWF molecule and the three chains comprising the GP Ib-IX complex, i.e., GP Ib(α), GP Ib(ß) and GP IX, have been determined, providing further insight into the structure-function relationships of these molecules. As a result, much recent investigation has been directed towards identifying small amino acid sequences in the primary chains of both GP Ib(α) and vWF which define the adhesive interaction. This review summarises current data concerning structure-function analysis of vWF and the GP Ib-IX complex and the putative identification of ligand- and receptor-binding domains involved in the interaction of vWF with the GP Ib-IX complex.

A variant of Glanzmann's thrombasthenia characterized by abnormal glycoprotein IIb/IIIa complex formation.

Glanzmann's thrombasthenia is a congenital bleeding abnormality characterized by absent platelet aggregation due to the failure of fibrinogen to bind to activated thrombasthenic platelets. In the majority of cases, this defect is caused by the absence or marked reduction of a specific fibrinogen-binding aggregation receptor, the GP IIb/IIIa complex. E.T., an 18-year-old female with a life-long history of bleeding and easy bruising, had the normal clinical features of Glanzmann's thrombasthenia. Surprisingly, sodium dodecyl sulphate-polyacrylamide gel electrophoresis of her platelets showed no apparent abnormality of the GP IIb/IIIa complex. Control platelets washed in the presence of 2 mM EDTA and control and patient platelets washed in the presence of 2 mM calcium ions showed normal reactivity with anti-GP IIb, anti-GP IIIa, and anti-GP IIb/IIIa complex specific monoclonal antibodies as evaluated by flow cytometry. In contrast, patient's platelets washed in the presence of 2 mM EDTA reacted with anti-GP IIb, anti-GP IIIa, but not with the complex-specific monoclonal antibodies. The increased susceptibility of the patient's GP IIb/IIIa complex to EDTA dissociation was confirmed by crossed immunoelectrophoresis (CIE). CIE analysis further indicated that the patient's GP IIb/IIIa complex did not bind fibrinogen. The combined results suggest that this patient has Glanzmann's thrombasthenia due to an abnormal association of the GP IIb/IIIa complex which results in the failure of the complex to bind fibrinogen.

Purification of botrocetin from Bothrops jararaca venom. Analysis of the botrocetin-mediated interaction between von Willebrand factor and the human platelet membrane glycoprotein Ib-IX complex.

Interaction of von Willebrand factor (vWF) with its platelet receptor only occurs in vitro in the presence of a modulator such as ristocetin. We have recently confirmed that the human platelet membrane glycoprotein (GP) Ib-IX complex is the receptor involved in the ristocetin-dependent binding of vWF by reconstitution with the purified components [Berndt, M.C., Du, X., & Booth, W.J. (1988) Biochemistry 27, 633-640]. We have now developed a similar solid-phase reconstitution assay using an alternate modulator, botrocetin, for the competitive analysis of functional domains in both vWF and the GP Ib-IX complex. Botrocetin was purified from Bothrops jararaca venom by ammonium sulfate fractionation and subsequent DEAE-cellulose and hydroxylapatite chromatography. The purified protein was a 25-kilodalton (kDa) disulfide-linked dimer with apparent subunit molecular weights of 14,000 and 14,500. Binding studies with immobilized botrocetin demonstrated that botrocetin bound to vWF and to a 52/48-kDa region of vWF that contains the GP Ib binding domain, but not to glycocalicin, a proteolytic fragment of GP Ib that contains the vWF binding site. Binding of 125I-labeled vWF to GP Ib-IX complex coated beads and to platelets was strictly botrocetin-dependent with half-maximal binding at a botrocetin concentration of congruent to 0.27 microM. Botrocetin-dependent binding of vWF was specific, saturable, and comparable to that observed with ristocetin. An anti-vWF monoclonal antibody, 3F8, inhibited ristocetin- but not botrocetin-dependent binding of vWF, suggesting the presence of distinct ristocetin and botrocetin modulator sites on vWF. The botrocetin reconstitution assay was at least an order of magnitude more sensitive than the corresponding ristocetin assay for the competitive analysis of functional domains on both vWF and the GP Ib-IX complex and has confirmed the localization of the vWF-binding domain to the 45-kDa N-terminal region of GP Ib.

Cross-linking of a monomeric 39/34-kDa dispase fragment of von Willebrand factor (Leu-480/Val-481-Gly-718) to the N-terminal region of the alpha-chain of membrane glycoprotein Ib on intact platelets with bis(sulfosuccinimidyl) suberate.

A 39/34-kilodalton (kDa) monomeric dispase fragment of von Willebrand factor (vWF) has been purified by heparin affinity chromatography. Detailed structural analysis of the individual 39- and 34-kDa fragments indicated that they had identical amino acid sequences extending from Leu-480/Val-481 to Gly-718 with an intramolecular disulfide bond between Cys-509 and Cys-695. In addition to the binding site for heparin, the 39/34-kDa fragment also contained binding sites for collagen and for platelet membrane glycoprotein (GP) Ib. Unlike native vWF, the 39/34-kDa fragment bound to GP Ib without the requirement for a modulator but showed increased binding in the presence of botrocetin. The 39/34-kDa vWF fragment was cross-linked to intact human platelets by using the membrane-impermeable, homobifunctional cross-linking reagent bis(sulfosuccinimidyl) suberate. Two distinct cross-linked species of similar molecular weight (220/200 kDa, nonreduced; 190/175 kDa, reduced) were identified by SDS-polyacrylamide gel electrophoresis and autoradiography, consistent with the cross-linking of the 125I-labeled 39/34-kDa vWF fragment to GP Ib. The formation of these cross-linked species was enhanced 1.5-2.5-fold in the presence of the modulator botrocetin. The platelet membrane protein involved in cross-linking was shown unequivocally to be GP Ib since (i) neither cross-linked species was formed with Bernard-Soulier syndrome platelets, which genetically lack the GP Ib-IX complex, (ii) both cross-linked species were specifically immunoprecipitated by anti-GP Ib polyclonal and monoclonal antibodies, and (iii) the formation of the cross-linked species was completely inhibited only by those anti-GP Ib-IX complex monoclonal antibodies that inhibited vWF-GP Ib-IX complex interaction. Proteolysis of cross-linked platelets with endoproteinase Lys-C, which preferentially cleaves off the N-terminal peptide domain on the alpha-chain of GP Ib, indicated that the 39/34-kDa vWF fragment was cross-linked exclusively to this region of the GP Ib-IX complex.

Bernard-Soulier syndrome.

Bernard-Soulier syndrome (BSS) is a rare autosomal bleeding disorder characterized clinically by prolonged skin bleeding time, normal clot retraction and thrombocytopenia with large and morphologically abnormal platelets, and biochemically by the absence of platelet membrane glycoproteins (GP) Ib, V and IX. GP Ib and GP IX exist in the platelet membrane as a heterodimer complex which acts as the major receptor mediating platelet adhesion to blood vessel subendothelium. Studies with BSS platelets have proved particularly rewarding in the investigation of the GP Ib-IX complex as a multifunctional receptor protein. The transmembrane complex contains binding domains for von Willebrand factor, thrombin, fibrin and quinine/quinidine drug-dependent antibodies as well as an attachment site on the cytoplasmic side of the membrane for a platelet cytoskeleton. In addition, the internal segment of the beta-chain of GP Ib contains a cyclic AMP-dependent protein kinase-associated phosphorylation site which appears to regulate platelet reactivity. Limited proteolytic cleavage of the complex, in particular the GP Ib alpha-chain, has allowed immunological and functional characterization of three distinct domains; a 45 kDa segment at the N-terminal end of the alpha-chain of GP Ib, which contains binding sites for von Willebrand factor and thrombin, a 90 kDa highly glycosylated region of GP Ib alpha and a membrane-associated region consisting of the remnant of GP Ib alpha disulphide-linked to GP Ib beta and non-covalently-complexed with GP IX. This membrane-associated region contains the antigenic epitope(s) for quinine/quinidine drug-dependent antibodies. It is highly probable that the future study of platelets from patients with the Bernard-Soulier syndrome will further clarify the role of the GP Ib-IX complex in platelet physiology.

Drug-mediated thrombocytopenia.

Thrombocytopenia due to drug-dependent antibodies most frequently occurs with quinine/quinidine and with heparin. Considerable evidence has accumulated about the mechanism of action of quinine/quinidine-induced antibodies but less is known about the effect of heparin. Although there is controversy, it is likely that the action of quinine/quinidine-induced antibodies follows a loose association between drug and platelet with antibodies acting independently of the Fc receptor. There is strong evidence that the complex of glycoprotein Ib and glycoprotein IX, absent in the Bernard-Soulier syndrome, provides the binding site for quinine/quinidine-dependent antibodies. It also appears that the two glycoproteins must be present in complex form for antibody binding to occur. There is some heterogeneity of quinine/quinidine-dependent antibodies since there are reports of a proportion of patient antibodies reacting with other membrane determinants or acting independently of the drug. Heparin-induced thrombocytopenia may be the consequence of a direct effect, or a more serious condition associated with thrombosis may occur when heparin-dependent antibodies are formed. The mode of action of these antibodies and the nature of their antigenic determinants remain unclear. Recognition of heparin-associated thrombocytopenia is important so that serious bleeding or thrombotic sequelae can be forestalled.

Glycoprotein Ib and glycoprotein IX are fully complexed in the intact platelet membrane.

Two new murine monoclonal antibodies, AK 1 and SZ 1, reactive with the human platelet glycoprotein (GP) Ib-IX complex have been produced by the hybridoma technique. Both AK 1 and SZ 1 immunoprecipitated the GP Ib-IX complex from Triton X-100-solubilized, periodate-labeled platelets. With trypsinized, labeled platelets, AK 1, SZ 1, and FMC 25 (epitope on GP IX) immunoprecipitated a membrane-bound proteolytic fragment of the GP Ib-IX complex consisting of GP IX and an congruent to 25,000 mol wt remnant of the alpha-chain of GP lb disulfide-linked to the beta-subunit. Unexpectedly, although AK 1 and SZ 1 immunoprecipitated purified GP Ib-IX complex, neither antibody immunoprecipitated the individual components of this complex, GP Ib or GP IX. When GP Ib and GP IX were recombined, however, AK 1 and SZ 1 again immunoprecipitated the reformed complex, strongly suggesting that both antibodies were recognizing an epitope present only on the intact complex. Cross-blocking studies indicated that AK 1 and SZ 1 recognized a very similar or identical epitope that was proximal to the epitope for FMC 25. Both AK 1 and SZ 1 bound to a similar number of binding sites (congruent to 25,000) on intact platelets as monoclonal antibodies directed against either GP lb or GP IX. The combined data suggests that GP lb and GP IX are fully complexed in the intact platelet membrane.

A murine antiglycoprotein Ib complex monoclonal antibody, SZ 2, inhibits platelet aggregation induced by both ristocetin and collagen.

A new monoclonal antibody (MoAb), SZ 2, reactive with the human platelet glycoprotein Ib complex has been produced by the hybridoma technique. SZ 2 immunoprecipitated the components of the glycoprotein Ib complex, glycoprotein Ib and glycoprotein IX, from Triton-X-100-solubilized, periodate-labeled platelets. Western blot analysis indicated that the epitope for SZ 2 was on the alpha-subunit of glycoprotein Ib. Scatchard analysis of SZ 2 binding to formaldehyde-fixed, washed platelets revealed a single class of binding sites with Kd = 6.6 +/- 3.3 X 10(-10) mol/L and 15,200 +/- 4,100 binding sites per platelet (mean +/- SD, n = 10). Intact antibody and its purified (Fab')2 fragments not only inhibited the ristocetin-dependent binding of von Willebrand factor to platelets and ristocetin-induced platelet agglutination but also inhibited platelet aggregation induced by Type I collagen and platelet-activating factor (PAF). SZ 2 inhibited platelet serotonin and beta-thromboglobulin release in response to these stimuli and also platelet thromboxane A2 formation in response to ristocetin and collagen. SZ 2 was without effect on platelet aggregation or release in response to other platelet stimuli such as ADP, thrombin, or arachidonic acid. The inhibition by SZ 2 of collagen- and PAF-induced platelet aggregation is surprising in that Bernard-Soulier syndrome platelets, which lack the glycoprotein Ib complex, respond normally to both these stimuli. SZ 2 was unreactive toward Bernard-Soulier syndrome platelets, as evaluated by fluorescence-associated cell sorting, and had no effect on the collagen- and PAF-induced aggregation of Bernard-Soulier syndrome platelets. The combined results suggest that the inhibition by SZ 2 of collagen- and PAF-induced aggregation of normal platelets is steric and are consistent with the glycoprotein Ib complex and the platelet collagen and PAF receptor(s) being adjacent in the human platelet plasma membrane.

Heparin-induced thrombocytopenia: further studies of the effects of heparin-dependent antibodies on platelets.

Studies were performed with the sera of five patients with heparin-induced thrombocytopenia. Each patient serum was found to cause heparin-dependent aggregation of normal and Bernard-Soulier syndrome platelets but not Glanzmann's thrombasthenia or fixed normal platelets. This antibody effect was blocked by inhibition of both aerobic and anaerobic metabolism and an increase in intraplatelet c-AMP with prostaglandin E1. These findings suggest that the antibody-induced platelet aggregation was not a passive agglutination of cells but an active process requiring metabolic energy and membrane glycoproteins IIb/IIIa but not glycoprotein Ib. In addition, platelet aggregation by four patient sera was inhibited by aspirin or apyrase indicating a prostaglandin synthesis and ADP-dependent mechanism and the reaction could be suppressed by dipyridamole alone. However, platelet aggregation induced by the serum of one patient was only inhibited partially by the combination of aspirin and apyrase suggesting mediation of the antibody effect in part by a pathway independent of extracellular ADP and prostaglandin synthesis and this antibody reaction could only be completely suppressed by a combination of aspirin and dipyridamole. Heterogeneity exhibited by these antibodies may influence the choice and effectiveness of antiplatelet therapy in heparin-induced thrombocytopenia.